This work is designed to understand the force-gated transduction channels in the hair cells of the inner ear, which perform the fundamental conversion of sound into a neural signal. The two aims of the research are to learn how these channels are positioned to respond to force, and also how they produce a feedback force that amplifies the incoming sound. Each hair cell has a bundle of actin-based stereocilia arranged with increasing heights; each stereocilium of a cell extends a filamentous 'tip link' to the next taller stereocilium. Movement of the bundle tightens tip links; they in turn pull open force-gated ion channels that open to depolarize the cell. Yet it is not clear where the transduction channels are in relation to the tip links, and so we cannot begin to understand in detail the mechanical linkage that activates them. We will use three new optical techniques to locate the transduction channels. One is swept-field confocal microscopy, which offers temporal resolution of milliseconds. A second is 2P-STED microscopy, a newly developed super-resolution microscopy that offers spatial resolution three-to-five-fold better than conventional light microscopes. Both will localize transduction channels with the use of dyes that detect Ca2+ entering through the channels. A third is STORM microscopy, another super-resolution technique which can observe the individual tip links that are connected to the channels and detect their angle and polarity. The opening and closing of transduction channels involves protein movements on the scale of a few nanometers, but these movements can move the entire hair bundle of a hair cells by tens of nanometers. Just milliseconds after transduction channels open, the Ca2+ entering through them causes them to close again, a process termed fast adaptation. Channel closure terminates the inward current, observed with a patch-clamp amplifier, but it often produces a fast backwards movement of the hair bundle, observed with a glass fiber probe. Movements associated with channel closure, although minute, have been proposed to underlie an active mechanical feedback in the mammalian cochlea that amplifies the incoming sound by 100-fold or more, and that creates an exceptionally sharp frequency tuning which enables sensory discrimination of tones. Yet it is not known how the basic force production works, i.e., how Ca2+ closes channels. We will use flexible glass fiber probes to stimulate hair bundles and to record their movement, and will control Ca2+ in the hair bundle directly by photolytically releasing it with a pulsed laser. Ca2+ will be released with the bundle biased to different positions, to map out the dependence of Ca2+-induced movement on position. The results will be compared to the predictions of each of four different models for Ca2+ action. A clear understanding of how Ca2+ produces fast adaptation can be incorporated into models for how cochlear amplification works.